The present invention relates generally to apparatus and methods for separating individual components from a mixture; more particularly, it relates to apparatus and methods which separate such components by the use of electrophoresis.
The term electrophoresis is generally used to describe the migration of a suspended particle in an electric field. The principles of electrophoresis have long been known but have only more recently been used extensively in separations technology.
Of particular importance is the use of electrophoresis to separate materials of biological origin for examination, particularly molecules of high molecular weight such as, for example, proteins, enzymes, nucleic acids, complex lipids and other carbohydrates. It is highly desirable when separating such materials to cause as little damage to or alter the molecules so that their properties are not changed significantly for analysis. The use of electrophoresis provides one significant means of accomplishing this.
There are apparatus available for performing separation by electrophoresis. Most generally comprise two electrodes with a separation chamber having a separating matrix therein, usually a gel, interposed between the electrodes. The components of the mixture migrate at different flow rates through this matrix under the influence of an applied electric field, with the different components emerging from the separating chamber one after the other.
These components are collected for sampling and analysis usually by one of two methods. The apparatus may be allowed to run for a specified period of time after which the components are collected directly from the matrix, or the components are allowed to flow completely through the matrix and into an elution chamber where they carried out by a flowing buffer solution.
Originally the separating matrix utilized in electrophoresis apparatus was a free solution (free-flow electrophoresis); however, the free-flow systems were impaired greatly by thermal convection and sedimentation within the free-flow medium. This technology has now been largely supplanted by the use of supporting mediums, particularly gels. The purpose of the supporting medium is to decrease the flow currents within the medium so that the separated components remain as sharp zones with maximum resolution between these zones. The supporting mediums are preferably chemically inert during the electrophoresis operations, generally uniform in their properties and able to be readily prepared and reproduced. Two of the more common supporting mediums are polyacrylamide gels and aragose gels.
As previously mentioned, a great variety of apparatus exist for carrying out electrophoresis. Most only significantly differ in their construction of the separating chamber. Two basic separating chamber designs currently prevail: cylindrical gel columns and slab gel designs. For example, U.S. Pat. No. 4,111,785 discloses a particular electrophoresis apparatus utilizing a cylindrical gel column separating chamber (the apparatus is currently manufactured and sold through Bethesda Research Laboratories, Gaithersburg, Md.). Also, U.S. Pat. Nos. 4,088,561 and 4,130,471 disclose particular electrophoresis apparatus utilizing slab gel separating chambers.
These apparatus, however, suffer from many shortcomings. For example, the cylindrical gel columns only provide limited resolution for low concentration components and are subject to overheating. Overheating of the gel column results in loss of stability and uniformity within the separating media, distortion between the zones of separation and loss of resolution within the system. Because of this overheating problem, the size of the cylindrical gel columns and the amount of power supplied thereto is limited. Further, cylindrical gel columns are generally cumbersome, difficult to assemble, complex to operate and somewhat unsafe during operation. Slab gel columns suffer from these same shortcomings plus they require a large volume of buffer solution to be used, further diluting the components and lowering the resolution of the system.
It is therefore an object of the present invention to provide an electrophoresis apparatus and method of operation for not only separating individual components from a mixture, but also concentrating those components as they are separated within the apparatus to improve the resolution of the apparatus and quality of results therefrom.
It is a further object of the present invention to provide an electrophoresis apparatus having improved heat exchange capability within the gel column so that higher power levels can be utilized within the apparatus without loss of resolution or decreased quality of results.
It is a still further object of the present invention to provide an electrophoresis apparatus of relatively simple construction, which is uncomplicated to use, and which is also safer to operate.